Table 3.

Summary of findings for each leukocyte population examined for each condition tested and subsequent recommendations for implementing the TP1 assay in a multicenter clinical trial.

Cell Type	Parameter	Antibody Staining Panel Storage at 4°C (0–8 weeks)	Time between blood draw and staining (2–24 hours)	Stained samples shipped at 4°or −80°C (compared to 2 day storage at same temperature)	Storage of stained samples at 4°or −80°C (3– 120 days)b
Granulocytes	Cells/µl whole blood	No Effect	No Effect	4°C causes decrease; No effect at −80°C	No Effect
CD3+ lymphocytes	Cells/µl whole blood	No Effect	No Effect	4°C causes decrease; No effect at −80°C	No Effect
CD4+ T cells	Cells/µl whole blood	No Effect	No Effect	4°C causes decrease; No effect at −80°C	No Effect
CD8+ T cells	Cells/µl whole blood	No Effect	No Effect	4°C causes decrease; No effect at −80°C	No Effect
B cells	Cells/µl whole blood	No Effect	No Effect	4°C causes decrease; No effect at −80°C	Decrease at 13 days when stored at 4°C ; No effect at −80°C
mDC	Cells/µl whole blood	No Effect	Increase at 24 hours	4°C causes decrease; No effect at −80°C	Decrease at 13 days when stored at 4°C ; Slight decrease beginning at 23 days at −80°C
pDC	Cells/µl whole blood	No Effect	No Effect	4°C causes decrease; No effect at −80°C	Decrease at 13 days when stored at 4°C ; Slight decrease beginning at 23 days at −80°C
CD56bright NK cells	Cells/µl whole blood	No Effect	No Effect	4°C causes decrease; No effect at −80°C	No Effect
CD56dim NK cells	Cells/µl whole blood	No Effect	No Effect	4°C causes decrease; No effect at −80°C	No Effect
CD56-NK cells	Cells/µl whole blood	---a	Increase at 24 hours	4°C causes decrease; No effect at −80°C	No Effect
Classical monocytes	Cells/µl whole blood	No Effect	Decrease at 24 hours	4°C causes decrease; No effect at −80°C	No Effect
Intermediate monocytes	Cells/µl whole blood	No Effect	Vary beginning at 4 hours	4°C causes decrease; No effect at −80°C	Increase at 13 days when stored at 4°C; No effect at −80°C
Non-classical monocytes	Cells/µl whole blood	No Effect	Vary beginning at 4 hours	4°C causes decrease; No effect at −80°C	No Effect
CD86high HLA-DRhigh classical monocytes	Frequency of Parent	No Effect	Increase at 24 hours	No effect at either temperature	Increase at 13 days when stored at 4°C ; Decrease beginning at 13 days at −80°C
CD86high HLA-DRhigh intermediate monocytes	Frequency of Parent	No Effect	Vary beginning at 4 hours	No effect at either temperature	Decrease beginning at 13 days for both temperatures
CD86high HLA-DRhigh non-classical monocytes	Frequency of Parent	No Effect	Vary beginning at 4 hours	No effect at either temperature	Decrease beginning at 13 days for both temperatures
Protocol Recommendation		Prepare antibody staining panel in large batches and store for up to 8 weeks at 4°C.	Stain blood within 4 hours of blood draw if interested in monocyte populations and 8 hours for consistent data on DC or NK cell populations.	Ship samples overnight on dry ice at −80°C.	Store samples at −80°C and collect them on the flow cytometer within 2 weeks of staining if interested in DC concentrations or monocyte activation. A reduction in the CD86 PE Cy5 MFI indicates that decreased activation percentages are likely due to loss of CD86 PE Cy5 staining.

^aCD56- NK cell counts were not able to be reliably gated due to very low frequency in the participant tested in this experiment.

^bSamples were only stored at 4°C up to 13 days beca use preliminary testing showed that gating was not possible past this point due to increased autofluorescence and reduced separation in some channels.